Induction of a parasitemia and preventing neuroinflammation by binding cholinergic receptors in apicomplexan infections using levamisole HCl

ABSTRACT

Equine protozoal myeloencephalitis (EPM) is the most commonly diagnosed infectious neuro-degenerative in horses. Important advances have been made in the biology of  S. neurona  but little attention has been paid to the inflammatory component of disease although neuroinflammation is present in all definitively diagnosed cases of EPM. Polyneuritis equi (cauda equina neuritis) of horses is a rarely recognized non-infectious, chronic relapsing, peripheral neurodegenerative disorder in horses. We propose that polyneuritis equi may be associated with  Sarcocystis neurona  infections. We demonstrate the presence of serum antibodies against myelin proteins and  S. neurona  surface antigens in 78% (n=55) ataxic horses with a presumptive diagnosis of EPM. Serum antibodies against  S. neurona  were detected in a horse with histopathologically confirmed polyneuritis equi, also sarcocysts were observed by histopathology. A clinical response to levamisole HCl treatment in ataxic horses may support cytokine mediated mechanism of the neuropathy.

INTRODUCTION

Equine protozoal myeloencephalitis (EPM) is the most commonly diagnosed infectious neuro-degenerative in horses. Important advances have been made in the biology of S. neurona but little attention has been paid to the inflammatory component of disease although neuroinflammation is present in all definitively diagnosed cases of EPM. Histopathologic examination of horse with EPM overwhelmingly reveals central nervous system (CNS) inflammation without the associated CNS presence of protozoa. Despite this finding, it remains dogma that the clinical signs of EPM are due to presence of parasites in the CNS and that the area of parasitization determines the signs observed. (1) It is likely that the signs are due to a combination of parasitization and inflammation. Polyneuritis equi (cauda equina neuritis) of horses, is a rarely recognized non-infectious, chronic relapsing, peripheral neurodegenerative disorder in horses. We propose that polyneuritis equi may be associated with Sarcocystis neurona infections. We demonstrate the presence of serum antibodies against myelin proteins and S. neurona surface antigens in ataxic horses with a presumptive diagnosis of EPM. Serum antibodies against S. neurona were detected in a horse with histopathologically confirmed polyneuritis equi, also sarcocysts were observed by histopathology. A clinical response to levamisole HCl treatment in ataxic horses may support cytokine mediated mechanism of the neuropathy. (2)

Infectious causes of neurodegenerative disease are difficult to identify pre-mortem because there are no pathognomonic diagnostic tests for each condition. Presence of sarcocystiasis (evidence of current or prior infection) is common in horses while sarcocystosis (presence of active infection) is rare. Exposure to S. neurona is common but it does not indicate when the exposure occurred. With a live clinically ill animal, serial measurements are used to track the immune response to active infection, but a single measurement gives no indication of the underlying trend so with just a single measurement, an active infection cannot be distinguished from residual antibody from previous infection.

A large number of clinically normal horses have circulating antibodies to S. neurona. Seropositive horses can have neurologic disease that is unrelated to S. neurona. The definitive diagnosis of infectious neurodegenerative diseases due to herpes virus encephalomyelitis (EHV), neuroborreliosis (Lyme), EPM, and polyneuritis equi rely heavily on ruling out other causes of the clinical signs. So, horses with disease due to EHV, Lyme, and polyneuritis equi may be S. neurona seropositive.

There are no antibody tests for diagnosis of acute EHV. (3) Serologic testing has limitations in confirming a diagnosis of EHV infection in an individual horse, testing of paired serum samples for incontact horses is recommended providing indirect evidence that EHV is the etiologic agent for a diseased individual. Treatment of EHV is palliative.

One may suspect Lyme disease in regions of the country that have ticks that harbor B. bugdorferi. Signs of borreliosis include generalized stiffness, hyperesthesia, shifting-limb lameness, muscle atrophy, chronic weight loss, and poor performance. Rare Lyme-associated clinical signs include neuroborreliosis, pseudolymphoma, and uveitis. Detection of antibody against B. bugdorferi is not definitive, is not evidence of active or incipient Lyme borreliosis, nor an indication of the need for treatment. (4) Further, horse infections with B. bugdorferi and S. neurona are not mutually exclusive. Detection of anti-equine myelin P2 antibodies in horses with polyneuritis equi was first reported in 1981. (5) (6)

The pathology and clinical finding of polyneuritis equi were initially described in 1897 in Germany and has since been reported in Europe and the United States. (7) Polyneuritis equi is a progressive, chronic, and often relapsing-remitting disease that affects the sacral and coccygeal nerves leading to paralysis of the tail, rectum, and bladder with loss of sensation in the sacral dermatomes with surrounding hyperesthesia. This disease is often accompanied with cranial nerve paralysis, particularly the facial and trigeminal nerves. The cranial deficits may precede clinical signs associated with involvement of the sacral and coccygeal nerves. Clinical signs of polyneuritis equi can include behavior changes, ataxia, proprioceptive deficits, and possibly a side-winding gait. (8) (7) (9) Antemortem diagnosis of polyneuritis equi by histopathology and circulating myelin protein serum antibodies have been described. (8) (6) Rostami and Gregorian indicated that anti-myelin serum antibodies may differentiate a T cell mediated peripheral neuritis from clinical signs associated with conditions that induce CNS lesions in horses. The etiology of polyneuritis equi is unknown but association with equine herpesvirus has been dismissed because histopathological lesions following experimental and naturally occurring EHV-1 infections are different from those of polyneuritis equi. The EHV-1 virus has not been isolated from horses affected with polyneuritis equi. An allergic neuritis in response to antigen released by trauma or infection, or a post infectious allergic neuritis, comparable in its pathogeneses and lesions to the human Guillain-Barre syndrome and its laboratory model, experimental allergic (autoimmune) neuritis (EAN) is likely. (7) Similar associations between anti-myelin proteins and disease are made in humans with multiple sclerosis (MS).

The prevailing theory as to the cause of polyneuritis equi is that it is immune mediated. Sarcocystis neurona infections can result in signs of involvement of cauda equina thus mimicking polyneuritis equi. A causal relationship between S. neurona infections and polyneuritis equi has not been established.

We hypothesize that immune reactions to S. neurona infections may initiate inflammation that can develop into polyneuritis equi. Untreated horses presenting with signs of polyneuritis equi may have serum antibodies against equine neurogenic myelin protein P2, (MP2) and S. neurona. (8) (6) (5) (7) (9) There is evidence that levamisole can alter immune mediated disease in humans and the clinical course of MS. (10) Levamisole HCl was investigated for the treatment of MS in people. (10) Levamisole HCl inhibits the production of IL6, a pro-inflammatory cytokine, in mice, dogs, and humans. (11) (12) (13) (14) A clinical response to levamisole HCl in horses with chronic, relapsing signs of EPM was demonstrated. (2)

MATERIALS AND METHODS

Sarcocystis neurona SAG 1, 5, 6 ELISA was performed as previously reported. (18) Based on proteomics data a 22 amino acid peptide (myelin protein peptide, MPP) that corresponds to the neuritogenic site associated with peripheral demyelinating disease, FIG. 1, was synthesized (United BioSystems, Herndon Va.). The MPP peptide was used in an indirect ELISA as described by Fordyce et al. Antigen coating dilution was optimized using a checkerboard titration with sera positive for anti-myelin antibodies (equine). We analyzed sera from 172 horses by indirect MPP ELISA using a modification of the Fordyce procedure by using MPP coated and blocked plates. The plates were incubated for 2 hours at 37 C, washed and alkaline phosphatase conjugated anti-horse whole molecule at 1:3000 was added and incubated for 30 minutes at 37 C. The plates were washed and reacted with PMPP for 15-30 minutes. Plates were read on an ELISA plate reader, the reciprocal of the last dilution positive (OD 0.518 or greater) was recorded at the titer. A positive titer was a value ≧8.

FIG. 1.

Amino acid sequence of MPP with putative epitope homology with S. neurona and IL6 by colored lines beneath the sequence. The broken gray line above the sequence indicates non-disease inducing peptide and the solid black line indicates the putative T cell epitope for the clinical and histological disease.

FIG. 2.

Areas of amino acid homology with S. neurona and equine IL6

Epitope Amino acid sequence SnSAG4 NTEISFKLG SnSAG6 EFEETTAD SFKLG FEET TTAD

Horse 10523

S. neurona One neurologically abnormal 12 year old Warmblood gelding (#10523) with serum antibodies against S. neurona SAG 1, 5, 6 also had serum antibodies against MPP (titer 64) before treatment. The presence of antibodies for both S. neurona and equine myelin protein are useful diagnostic indications of the conditions, but do not measure the severity of the condition, therefore a positive/negative value and not end point titers were used for our analysis. The horse was diagnosed with peripheral myositis by histopathology (Oregon State University Veterinary Diagnostic Laboratory). Muscle Sarcocystis cysts were confirmed by histopathology.

Screen Sera for MPP Antibodies by ELISA

S. neurona Sera from horses (172) with clinical signs of EPM and 10 neurologically normal horses were tested by MPP ELISA.

One hundred seventy two sera were tested for serum MPP antibodies by MPP ELISA. Seventy seven sera of 172 (45%) were positive for antibodies (MPP ELISA titer ≧8) establishing that MPP reactive antibodies are detected in some horses. The presence of antibodies for both S. neurona and equine myelin protein are useful diagnostic indications of the conditions, but do not measure the severity of the condition, therefore a positive/negative value and not end point titers were used for our analysis. Pooled sera from positive horses were used as a positive control for each MPP ELISA.

Fifty Five Sera from Ataxic Horses, Pre and Post EPM Treatment, were Tested for SnSAG 1, 5, 6 and MPP Antibodies

Fifty five neurologically abnormal horses were tested for serum antibodies against S. neurona (SAG 1, 5, 6), C-reactive protein, and MPP by ELISA. Sera was submitted by attending veterinarians that were familiar with the horses and assigned a gait assessment score (GAS) of 1-4.

Gait assessment scores were assigned using the following criteria:

GAS 0 no abnormalities; GAS 1 gait deficit jus detected at a walk, drags limbs, circumducts limbs(s) on tight circling, mild weakness noted on tail pull-easily pulled off track, but regains normal stride in 1-2 steps; GAS 2 gait deficit easily detected and exaggerated by backing, turning or neck extension, swaying at walk, displays a wide based stance after tight circling, weakness on tail pull-easily pulled off track, and dos not return to normal stride for more than 3 steps. A GAS 3 was a gait deficit prominent on walking, turning or neck extension, unable to perform tight circles without falling backwards, weakness on tail pull easily pulled off track and almost falls, doesn't regain normal stride. A GAS 4 is a horse that stumbles often, tripping spontaneously during exam, falls spontaneously and will fall upon backing or tight circles, weakness on tail pull pulled off track and could fall. A GAS of 5 is a recumbent horse that is unable to rise without assistance, and leans on a wall for support, spins to regain balance and may have seizures. Thirty one (56%) of horses were seropositive for S. neurona and twenty-four, 44% were seronegative. The majority of horses were seropositive for MPP, 43 (78%) were positive. Twelve horses (22%) were MPP seronegative. These data indicate that the majority of horses have a peripheral neuropathy associated ataxia. Fifty-one sera were evaluated for C-reactive protein (CRP) by capture ELISA. A serum CRP concentration ≧10 was detected by capture ELISA in 66% (n=34) of the samples before treatment and a serum CRP concentration <10 was detected in 33% (n=17) of the samples.

TABLE 1 MPP+ (78%) MPP− (22%) S. neurona SAG+ (n = 31, 56%) 24 (56%) 7 (58%) S. neurona SAG− (n = 24, 44%) 19 (44%) 5 (41%)

The gait assessment scores assigned by a veterinarian, GAS, were evaluated for success of treatment by evaluating a change in gait (GAS) by the difference in score before and after treatment. Successful response to treatment was a (GAS) that was >0 and a treatment failure was a (GAS) that was ≦0. Successful response to treatment was evident in 23 (86%) of horses that were positive by MPP ELISA and 25 (89%) of horses that were negative by MPP ELISA. These data indicated that treatment with levamisole HCl can alleviate signs due to peripheral and central neuropathies associated with S. neurona.

TABLE 2 MPP+ MPP− (GAS) =0  4 (14%)  3 (11%) (GAS) >0 23 (86%) 25 (89%)

Discussion

Equine protozoal myeloencephalitis was suspected in horses with circulating S. neurona antibodies and an abnormal gait. Highly specific bioassay using surface antigens SAG 1, 5, and 6 are useful to detect serum antibodies in horses with sarcocystiasis. It is now recognized, from molecular analysis and population genetics, that SnSAG1, SnSAG5, and SnSAG6 surface antigens will predominate in the S. neurona strains that circulate in nature and infect horses. (1) Immune-mediated polyneuritis equi is associated with the presence of equine myelin protein 2 auto-antibodies. (5) (6) Serum antibodies against S. neurona and MPP were detected in a horse with histologically confirmed polyneuritis equi. We detected antibodies against MPP in 45% in the serum of ataxic horses, but not in normal horses. A slight majority of horses with antibodies against S. neurona are MPP positive in this study indicating that some of the clinical signs of EPM may be due to an autoimmune peripheral neuritis.

Myelin protein peptide is a logical choice for detecting anti-myelin antibodies in equine serum because the peptide contains the neuritogenic site for induction of autoimmune neuritis. Myelin proteins are largely conserved across species although equine myelin differs by a small increase in the number of amino acids. There is a large amount of myelin P2 protein in the (CNS) tissues of horses when compared to other species. It was demonstrated that peripheral and CNS derived myelin P2 in the horse is practically identical to other myelin P2 binding structures. (15) Induction of experimental autoimmune neuritis (EAN) by whole peripheral nerve myelin P2 (MP2) protein, or partial peptides derived from the whole protein, results in an inflammatory demyelinating disease in several species. (16) The induction of EAN with MP2 is dose dependent. (16) The neuritogenic site within the P2 protein was determined, a synthetic 26 amino acid peptide (SP-26) induces clinical and pathological signs of EAN in rats. A shorter synthetic peptide (SP 61-72), based on amino acids of MP2 are ineffective at inducing pathological disease.

Anti-MMP antibodies are expected to preferentially detect peripheral neuropathies. Rats immunized with SP-26 develop extensive inflammatory demyelinating changes in ventral roots and sciatic nerves, but no central nervous system changes. Further, there was a positive correlation between the clinical severity of EAN and the dose of SP-26 in immunized rats. Lymph node cells from SP-26-immunized rats generated reactive T cells specific for SP-26. (16) These data demonstrated a cellular immune response by T helper cells to SP-26. Rostami and co-workers concluded that the antigenic site for EAN induction in rats is located within the 53-78 amino acid residues and that SP-26 could contain an important T cell epitope (8-11 amino acids) for the induction of EAN. Rostami and coworkers showed that stimulating SP-26-specific neuritogenic T cell lines adoptively transferred EAN in naive Lewis rats. (17) They assert that at least one T cell epitope for the induction of EAN resides within the 53-78 residues of myelin P2 protein which may be the antigenic site for the disease process. (17) They also showed that the induced cellular immune response to the P2 protein was milder than induced responses to SP-26. It was interesting that the T cell line lost its proliferative activity to P2 protein activity, whereas SP-26 activity was not affected. Based on the foregoing it is logical to use the putative disease-inducing peptide to detect anti-myelin antibodies in horses with suspected peripheral neuropathy. The absence of MPP antibodies in some ataxic horses in this study may indicate that clinical signs are due to central neuroinflammation and not peripheral neuroinflammation.

A putative T cell epitope that is important in the induction of autoimmune polyneuritis is contained within the amino acid residues of MPP. Detection of serum antibodies to MMP may differentiate a T cell mediated peripheral neuritis from clinical signs associated with conditions that induce central nervous system lesions in horses. However, it would be possible to have both central nervous system and peripheral neurodegenerative disease associated with EPM in the same animal. Protozoa rarely remain in the CNS of horses that continue to exhibit progressive clinical signs of neurodegeneration leading to speculation that the pathogenesis of disease is initiated by parasites, but disease progression is not parasite dependent. One may speculate as to the relationship of protozoa-mediated inflammation and cytokine mediated encephalomyelitis by molecular analysis.

A molecular (BLAST, Basic Logical Alignment Tool NCBI) analysis of the amino acids 57-78 (MMP) of the SP-26 peptide against the Sarcocystis neurona NCBI data bank reveals homology with amino acids from surface antigens (SnSAG) 4, 5, 6, 1 of S. neurona and equine IL6. It was previously shown that at least one T cell epitope for the induction of EAN resides within the 53-78 residues of myelin P2 protein, the putative antigenic site for the disease process, but not within residues 61-72. An amino acid peptide 61-72 of myelin P2 protein did not induce histological or clinical EAN in rats. Surface antigen 6, 1, and equine IL6 are identical to amino acids 69-77, 71-74, and 74-77, respectively. Homology with the reactive antigenic sites for myelin protein associated autoimmune polyneuritis may have significance for the pathogenesis of EPM in horses. Further, our results support that IL6 may be the reactive T cell epitope in horses with autoimmune polyneuritis equi. Surface antigen SAG 4, a common Sarcocystis surface antigen, is identical to amino acids of MPP at residues 60-68 showing homology within the non-disease inducing 61-72 residues of myelin P2 protein. The surface antigen SnSAG 5 also is inside the non-reactive part of the peptide at residues 64-68. It remains to be determined if the three dimensional structure of myelin P2 protein grants significance for these antigens, or a combination of these antigens, in the inflammatory component of EPM. Sarcocystis neurona sheds major surface antigens during infection and it is possible that these S. neurona antigens react with T cell epitopes that induce neurodegenerative disease in horses. A possible mechanism of the neuroinflammatory component of EPM syndrome can be speculated if clinically ill horses have serum antibodies against SAG 1, 5, 6 and MPP as we demonstrated.

Supporting the association of the mechanism of disease with equine cytokine IL6 is the clinical response to levamisole HCl because levamisole HCl inhibits the production of IL6. A clinical response was apparent in 48 of 55 treated horses. It would be beneficial to differentiate horses with IL6 mediated peripheral neuropathies versus central neurodegenerative conditions because treatment duration may differ. Our data supports that some, but not all horses may have peripheral neuritis and at least one etiology of polyneuritis equi may be Sarcocystis neurona infections (Table 1). Because the presentation of autoimmune polyneuritis equi is chronic and relapsing it is expected that antibodies against MPP would be detected in conjunction with clinical signs. It was not determined how long MPP antibodies remain in the successfully treated horses. Our data indicates that levamisole HCl may treat both peripheral and central neuroinflammation associated with protozoal infections, the horses were treated with decoquinate and levamisole but decoquinate does not have a target molecule in horses. There was little difference between horses with and without serum MPP antibodies that show a positive response to treatment (Table 2) indicating another condition, central neuroinflammation, may be present in some horses. Levamisole HCl is potentially beneficial to alleviate clinical signs of peripheral neuritis equi associated with S. neurona infections, alleviating clinical signs in more than 85% of the treated horses (Table 2).

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1. A method for reproducing an Apicomplexan parasitemia in a mammalian host, said method comprising infecting an isolated susceptible cell homologous to said mammalian host with a merozoite stage of the Apicomplexan parasite; inoculating said infected susceptible homologous cell into said host; blocking the infection by administering a cholinergic agonist to the host; and monitoring said host for Apicomplexan parasitemia.
 2. A method of selecting and testing new drugs for treatment of Apicomplexan diseases in a mammalian host, said method comprising compounds that are cholinergic agonists
 3. The method of claims 1 and 2 wherein the Apicomplexan parasite is selected from Sarcocystis dasypus (syn. S. neumna), Sarcocystis neuron, Sarocystis falcatula, Toxoplasma gondil, Neopora caninum, Neospora hughesi, Samocystis cruzi, Sarcocystis spp., Eimeria spp. and Plasmodium spp.
 4. The method of claims 1 and 2 wherein the mammalian host is selected from the group consisting of humans, equines, bovines, canines, felines, birds, goats, sheep, mice, rats, guinea pigs, rabbits, and hamsters.
 5. The method of claim 1 wherein the susceptible homologous cell is selected from the mammalian host consisting of humans, equines, bovines, canines, felines, birds, goats, sheep, mice, rats, guinea pigs, rabbits, and hamsters.
 6. The method of claims 1 and 2 wherein the cholinergic agonist is selected from the group consisting of Levamisole, imidazothiazole, salts thereof; esters thereof and derivatives thereof.
 7. The method of claim 6 wherein said cholinergic agonist is administered by a route selected from the group consisting of oral, intranasal, topical, inhalation, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intratracheal injection, intrathecal injection and combinations thereof.
 8. The method of claim 2 wherein the dose of cholinergic agonist is between 0.001 and 100 mg/kg.
 9. The method of claim 8, wherein the cholinergic agonist is Levamisole HCl.
 10. The method of claim 9, wherein the dose of Levamisole HCl is between 0.1 and 10 mg/Kg.
 11. The method of claim 8 wherein the dose of Levamisole HCl is 1 mg/Kg.
 12. The method of claims 1 and 2 wherein the dosage form is selected from the group consisting of a powder, a solid, a liquid, a crystal, a pill, a tablet, a capsule, a thin film, a suspension, a paste, a cream, a gel, a liniment, a balm, a lotion, an ointment, and a skin patch. 